Process for producing an antigen for use in the diagnosis of malaria and product thereof



Patented May 10, 1949 PROCESS FOR PRODUCING AN ANTIGEN FOR USE IN THEDIAGNOSIS OF MALARIA AND PRODUCT THEREOF Anna Dean Dulaney, Memphis,Tenn., assignor to The University of Tennessee Research Corporation,Knoxville, Tenn, a corporation of Tennessee N Drawing. ApplicationSeptember 17, 1945, Serial No. 616,959

Claims.

This invention relates to the production of an improved antigen for usein the diagnosis of malaria.

Complement fixation tests have been developed to aid in routinelaboratory determination of malaria. They depend upon the use ofantigens ordinarily produced as saline extracts in known manner fromdried parasites ground with saline solution and successively frozen,thawed and centrifuged (Dulaney and Stratman-Thomas, J. Immun, 1940,Vol. 39, pp. 247-255) While this procedure has proven dependable forsuch tests, yet th opinion is justified that the test is more specificthan sensitive. Recognition of the diagnostic possibilities of the testemphasizes the need for a more sensitive antigen.

It is an object of the present invention to produce a highly activeantigen for the diagnosis of malaria by complement fixation test by asimplified procedure avoiding the conventional successive freezing andthawing of the blood cells for the extraction of the antigen portion.Thus the time and cost of the process are both reduced materially.

It is a further object of the invention to improve the procedure byearly separation of the parasite mass from the hemoglobin of the bloodcells. This purification is highly advantageous in facilitating theextraction of the mass.

A further object of the invention is to diminish the color of the finalproduct. This is highly important as it makes reading or interpretingthe test much easier and certain.

An added feature of the invention is a great increase in the quantity ofthe antigen obtained from a given source due to the use of an extractantdiffering from the conventional saline solution. Specifically such anextractant may be a phosphate buffer solution or a barbituate buffersolution.

Other objects of the invention and consequent advantages andimprovements will be evident from the following detailed description ofthe preferred manner of carrying out my invention.

To obtain a parasite mass for use in obtaining my antigen I infectedmonkeys heavily with malaria through inoculation with the organism knownas Plasmodium knowlesi. At the peak of development of the infection themonkeys were killed and the blood collected.

The erythrocytes were laked with distilled water, freeing the parasitesfor further treatment.

The parasites were washed with water and centrifuged to removenon-parasitic material as cornpletely as possible. The parasites werethen dried for subsequent use in the preparation of the antigen. Thisdrying puts the material in form for accurate determination ofquantities 2 for processing. It is otherwise difficult to measure thequantity of parasites being used or to proportion the extractingmaterial.

The above preparatory routine is mainly preliminary to the principalfeature of my invention and well known except in so far as the lakingstep which is a distinct departure from customary practice.

The parasites prepared as above and in either wet or dry form wereground with M/lO phosphate buffer solution of pH 7.8-8.0 prepared by theusual and well-known method such as is set out on pages 810 and 811 ofPractical Physiological Chemistry by Hawk and Bergeim, tenth edition,1931. While alternate freezing and thawing at room or refrigeratortemperatures may be employed, it is found that the selection of theextracting solution obviates the need for this routine. The preferredpractice as followed was to allow the preparation to stand at roomtemperature with frequent stirring for about one hour. Centrifugationyielded a deep brown opalescent supernate. The residue was repeatedlyextracted with fresh buffer solution until recovery was negligible.

The relative antigenic activity was determined by titration with astrongly positive malaria serum. It was found that the bufferpreparations were much more active than the usual saline extracts.

As a specific example of the improved process and to illustrate thecomparative values of this and the conventional saline extraction, two0.1 gram samples of the dried parasites were each ground with 10 cc. ofphosphate buffer and labeled (a) and (b). A third or control sample wasground with 10 cc. of 0.9 percent sodium chloride and labeled (0).

The sample (a) was given the usual treatment by alternate freezing andthawing (four times) then centrifuged, yielding a clear brown supernatewhich was removed and designated P-FT-l. The residue was mixed with afurther 10 cc. of phosphate buffer solution and the alternate freezingand thawing repeated. The supernate from centrifuging was almostcolorless and was designated P-FT-Z.

Two successive supernates P-FT-S and P-FI'4 respectively were obtainedin like manner. Both were practically colorless.

The second sample (b) was allowed to stand at room temperature withfrequent stirring for about one hour. On centrifugation a deep brownopalescent supernate (P-R-l) was obtained.

The residue was taken up in 10 cc. of buffer and stirred at intervalsfor a second extraction period of about an hour. The pale amboropalescent supernate was designated P-R-Z.

Two additional extractions were made yielding 3 faintly coloredpreparations P-R-3 and P-R-4 respectively. Thefinalresidu'e: wastaken upin cc. of bufier solution, left in therefrigerator overnight andcentrifuged to produce a supernate P-R-5.

For purposes of furthercomparison-the;sample (c) was frozen and thawedas was sample (a) v but using 10 cc. amounts of 0.9 per cent sodiumchloride solution and successiver-extractsfobtained (S-FT-l; S-FT-Z;S-FT-3-and SJ.T-=4 -respec-.

tively).

By titration against a strongly positive malaria serum diluted 1:2the:relative-antigenic activity of each of the above extracts was 1determined. A known negative serum and anticomplementary controls wereincluded. All tests with the negative serum and in the series containingno serum were negative.- The-highest dilution of each antigen giving a4-plusreaction with the positive serumwasrecorded. 'Thefollowing tablegives the antigen units per cubic centimeter observed for eachextraction ineach -of the three procedures.

It is apparent that the phosphate buffer preparations are much moreactive than the saline extracts and that the values are-obtainedin theearlier extractions.

'A- calculated total of 9600 antigenic units-was -obtained by fourextractions with phosphate bufier solution atroom temperature asagainstinventor and D. B. Morrison. In this article (page 324) it isstatedthatHighl fi tiveantigens may be I, prepared. by treating wet -.or.. dried.parasites with M/lO phosphate buffer of pH 7.88.0 From the abovedescription it will be evident that I have provided a material anddistinct improvement in the process of preparing an anti- "genforcomplement fixation tests for malaria.

The process is marked by simplification in routine .with consequentsaving in time and cost. ff'hefinal product is improved in color andrenders makingrthe tests more easy and certain.

-Finally ,thequantity of antigen recovered has :beensubstantiallyincreased and the antigen found to have greater sensitivity or activity.Other'incidental advantages are present and the invention is clearlysubject to variation in minor details and factors within" the scope ofthe appended-claims.

The present application is limited to the phos- -phate bufier andprocess. Thebarbiturate buffer and process constitute the subject-matterof divisional applicationfierial No. 64,696, filed December 10,1948.

' Whatis claimed-is:

l; The process of preparing an antigen for complement fixation tests formalaria from parasitized animal blood which consists .in laking theblood cells, grinding the parasite mass in M/lO 7200 units by fourfreezingsand-thawings in phosphate and 1400' uni-ts'by four freezingsandthawings in saline solution.

A'further example of'theimproved process was carried outbytreatingfresh'or driedparasites with N/lO barbiturate buffer solution (pl-18.5)at room'or refrigerator temperatures. but. other-" wise "in the sameproportions and conditions; as above described. 'Eithermethod yieldedactive antigens -.con-

'taining 320m 640 units per'cubi'c centimeter and additional quantitieson repeated extraction.

A-highly active antigen was prepared-byextracting the wet parasitesobtainedfrom-one monkey with barbiturate: buffer solution as aboveoutlined, first with -200 cc. andthenWith SO cc.

The combined extracts werecleaned-of salts by dialysis against distilledwater and-subsequently concentrated ina semipermeable-membrane at 5'C.to40 cc. On centrifuga-tion-a supernate of 1280 units of antigen-per" cc.was obtained.

The antigen produced -in the 'mannerherein described is characterizedbya marked increase in sensitivity aswill *be-evident=from the abovetabulated results of tests. ":"It therefore constitutes a novelproduct-of--materially---enhanced utility.

' Further details relating' to the preparation of the antigen and itsuse in carrying-:Qutrcomplement fixation tests for malaria-are to.--befound in the article-on thepreparation andproperties of: antigens-fromPlasmcdium-knowlesi published in the AmericanJournalof'TropicaliMedicine, September,=1944,-*-vo1-.:24;:pp.e323-326;-by the 1 phosphate buffer solution of pH7.8-8.0, allowing the mixture to standfor approximately one hour with:frequent agitation and separating the; antigen.

2..:*Ihe process of; preparing an,.antigen for x:complement fixation.tests for, malaria from the ::blood of animals infectedpwith Plasmodiumknowlesiuwhich consists in rlaking the blood cells,

grinding the parasite mass; in M/ lll phosphate -;buffer solutionzofpH7.8-8.0, allowing the; mixture to stand for approximately one hour withfrequent agitation and separating the antigen.

.43. "I he12process;of preparing an; antigen for complement-fixation;-tests for-malaria from the i' blood 'of. wanimals infected .-with 1Plasmcdium Jcnowlesi; which consists. l-aking the blood cells,

grinding the,-- parasites; mass mild-phosphate buffer solution of pH-7.8-8.0; allowing the mixt-ure to stand ion-approximately one hour withfrequent z-agitation; centrifuging to separate supernatant antigen;repeating .the extraction awith ='fresh.-.bufier' solution and combiningthe everal supernates.

4L. The process-otpreparing an. antigen for complement fixation tests'for- .malaria from the blood I of animals infected with, PlasmodiumJcnowlesz, which consists ingr-indingtheparasitecontaining mass in M/10phosphate buffensolution.-of pI-I- 'Z.8-8.0, allowingithemixture .tostand .for approximatelytone. hour. withJrequent. agitation andseparating .the antigen.

5.- .Anantigen for .complement fixation' tests :fOI'{-m2.13,1ia.-consisting ,of the extract :in,,M/ 10 phosphate buffer solution ,of-.-pH 7.8.-,8 .0 of the parasites from .the. .blocdof, an..animal infected with Plasmodium knowlesi.

.. 1 ANNA DEAN DULANEY.

E ENCE CIT The following references are of -record;,,- in the;:file.';of this; patent: H w-Chem;Abs.,.vol;.36; 1942; cols; 4.887and4888. cmpleme wfi t n human ma ria. y

Dulaney :et -al.. and Bufferp-recipitaticn tests for

